Bioconversion of renewable lignocellulosic biomass to a fermentable sugar that is subsequently fermented to produce alcohol (e.g., ethanol) as an alternative to liquid fuels has attracted the intensive attention of researchers since the 1970s, when the oil crisis occurred (Bungay, H. R., “Energy: the biomass options”. NY: Wiley; 1981; Olsson L, Hahn-Hagerdal B. Enzyme Microb Technol 1996, 18:312-31; Zaldivar, J et al., Appl Microbiol Biotechnol 2001, 56: 17-34; Galbe, M et al., Appl Microbiol Biotechnol 2002, 59:618-28). The production of sugars from lignocellulosic biomass materials has been known for some time, as has the subsequent fermentation and distillation of the sugars into ethanol. Much of the prior development occurred around the time of World War II when fuels were at a premium in such countries as Germany, Japan and the Soviet Union. These early processes were primarily directed to acid hydrolysis, which were complex in engineering and design, and were typically sensitive to small variations in the processes, such as to temperature, pressure and/or acid concentrations. A comprehensive discussion of these early processes is found in “Production of Sugars from Wood Using High-pressure Hydrogen Chloride”, Biotechnology and Bioengineering, Volume XXV, at 2757-2773 (1983).
The abundant supply of petroleum in the period from World War II through the early 1970s slowed ethanol conversion research. However, due to the oil crisis of 1973, researchers increased their efforts to develop processes for the utilization of wood and agricultural byproducts for the production of ethanol. This research was especially important for development of ethanol as a gasoline additive to reduce the dependency of the United States upon foreign oil production, to increase the octane rating of fuels, and to reduce exhaust pollutants as an environmental measure.
Concurrently with the “oil crisis,” the U.S. Environmental Protection Agency promulgated regulations requiring reduced lead additives. Insofar as ethanol is virtually a replacement of lead, some refineries have selected ethanol as the substitute for its capability of easy introduction into a refinery's operation without costly capital equipment investment.
The high pressure and high temperature gas saccharification processes developed decades ago continue to be improved. New and current research focuses greatly on enzymatic conversion processes, which employ enzymes from a variety of organisms, such as mesophilic and thermophilic fungi, yeast and bacteria, degrading cellulose into fermentable sugars. Uncertainty remains with these processes, mainly on their ability to be scaled up for commercialization and on the efficiency of ethanol production.
Cellulose and hemicellulose are the most abundant plant materials produced by photosynthesis. They can be degraded for use as an energy source by numerous microorganisms, including bacteria, yeast and fungi, which produce enzymes capable of hydrolysis of the polymeric substrates to monomeric sugars (Aro et al., 2001). Organisms are often restrictive with regard to which sugars they use, and this dictates which sugars are best to produce during conversion. As we approach the limits of non-renewable resources, we recognize the enormous potential of cellulose to become a major renewable energy resource (Krishna et al., 2001). The effective utilization of cellulose through biological processes can potentially overcome the shortage of foods, feeds, and fuels (Ohmiya et al., 1997).
Cellulases are enzymes that hydrolyze cellulose (beta-1,4-glucan or beta D-glucosidic linkages) resulting in the formation of glucose, cellobiose, cellooligosaccharides, and the like. Cellulases have been traditionally divided into 3 major classes: endoglucanases (EC 3.2.1.4) (“EG”), exoglucanases or cellobiohydrolases (EC 3.2.1.91) (“CBH”) and beta-glucosidases ([beta]-D-glucoside glucohydrolase; EC 3.2.1.21) (“BG”) (Knowles et al., 1987 and Shulein, 1988). Endoglucanases act mainly on the amorphous parts of the cellulose fiber, whereas cellobiohydrolases are also able to degrade crystalline cellulose.
Cellulases have also been shown to be useful in degradation of cellulose biomass to ethanol (wherein the cellulases degrade cellulose to glucose, and yeast or other microbes further ferment the glucose into ethanol), in the treatment of mechanical pulp (Pere et al., 1996), for use as a feed additive (WO 91/04673) and in grain wet milling Separate saccharification and fermentation is a process whereby cellulose present in biomass, e.g., corn stover, is converted to glucose and subsequently yeast strains convert glucose into ethanol. Simultaneous saccharification and fermentation is a process whereby cellulose present in biomass, e.g., corn stover, is converted to glucose and, at the same time and in the same reactor, yeast strains convert glucose into ethanol. Ethanol production from readily available sources of cellulose provides a stable, renewable fuel source.
Cellulases are produced by a number of bacteria, yeast and fungi. Certain fungi produce a complete cellulase system (i.e., a whole cellulase) capable of degrading crystalline forms of cellulose. A whole cellulase, especially one that is naturally occurring, is, however, not necessarily capable of achieving efficient degradation because it may not include all the components/activities required for this efficiency, for example, activities from each of the CBH, EG and BG classifications. (Filho et al., 1996). It is known that individual CBH, EG, and BG components alone do not bring about efficienct hydrolysis, but the combination of EG-type cellulases and CBH-type cellulases interact to more efficiently degrade cellulose than either enzyme used alone (Wood, 1985; Baker et al., 1994; and Nieves et al., 1995).
Cellulases are known in the art to be useful in the treatment of textiles, for enhancing the cleaning ability of detergent compositions, for use as a softening agent, for improving the feel and appearance of cotton fabrics, and the like (Kumar et al., 1997). Cellulase-containing detergent compositions with improved cleaning performance (U.S. Pat. No. 4,435,307; GB App. Nos. 2,095,275 and 2,094,826) and for use in the treatment of fabric to improve the feel and appearance of the textile (U.S. Pat. Nos. 5,648,263, 5,691,178, and 5,776,757, and GB App. No. 1,358,599), have been described.
Hence, cellulases produced in fungi and bacteria have received significant attention. In particular, fermentation of Trichoderma spp. (e.g., T. longibrachiatum or T. reesei) has been shown to produce a complete cellulase system capable of degrading crystalline forms of cellulose. Over the years, Trichoderma cellulase production has been improved by classical mutagenesis, screening, selection and development of highly refined, large scale inexpensive fermentation conditions. While the multi-component cellulase system of Trichoderma spp. is able to hydrolyze cellulose to glucose, there are cellulases from other microorganisms, particularly bacterial strains, with different properties for efficient cellulose hydrolysis, and it would be advantageous to express these proteins in a filamentous fungus for industrial scale cellulase production. However, the results of many studies demonstrate that the yield of expressing bacterial enzymes from filamentous fungi is low (Jeeves et al., 1991).
Soluble sugars such as glucose and cellobiose have many uses for the production of chemicals and biological products. The optimization of cellulose hydrolysis allows for the use of less enzymes and improved cost effectiveness for the production of soluble sugars.
An efficient conversion of lignocellulosic biomass into fermentable sugars is key to producing bioethanol in a cost-effective and environmentally-friendly way. To reduce energy and processing cost, particularly for distillation, the minimum ethanol concentration produced by a viable process should be at least 4% (w/v). Such an increased ethanol concentration can be achieved by processing substrates having high dry matter of solids. However a common problem associated with saccharifying a high dry matter biomass is the high viscosity of the slurry, resulting in a slurry that is not pumpable or requires large energy input during handling. When dealing with handling of high solids, problems such as 1) insufficient mixing with limited mass transfer, 2) increasing concentration of inhibitors, such as acetic acid, furfural, 5-hydroxymethyl furfural, phenolic lignin degradation, 3) production inhibition, such as glucose, cellobiose, ethanol, and 4) fermentation microorganism viability, will occur. High viscosity limits the dry substance level in the process, increasing energy and water consumption, reducing the separation efficiency, evaporation and heat exchange, and ultimately, the ethanol yield. Reduction of viscosity is therefore beneficial, and enzymes play a key role in breaking down the soluble/insoluble compounds causing high viscosity.
Studies to increase solid loading and/or reduce viscosity of saccharification processes have taken place. For example, a number of studies utilized fed-batch operations in order to increase the solids level in the biomass substrate loading. A gravimetric mixing reactor design was used, which allowed batch enzymatic liquefaction and hydrolysis of pretreated wheat straw at up to 40% solids concentration. This fed-batch strategy sequentially loads the biomass substrate or substrate plus enzymes during enzymatic hydrolysis in order to achieve hydrolysis of a large amount of substrate, a relatively low viscosity during hydrolysis, and a relatively high glucose concentration during the process. Alternatively, enzymatic pre-hydrolysis of a lignocellulosic biomass for a period of time at the enzymes' optimum temperature, e.g., 50° C., can be carried out to reduce the viscosity of the slurry, enabling pumping and stirring. The decrease in viscosity during pre-hydrolysis makes the subsequent fermentation or SSF possible.
Despite the development of numerous approaches, there remains a need in the art for additional ways to reduce viscosity and improve yield of desirable fermentable sugars.
All references cited herein, including patents, patent applications, and publications, are incorporated by reference in their entirety.